The experiments conducted to determine the heat resistance of Bacillus megaterium ATCC 6458 at 90 and 100 C were completed. Estimates from replicate experiments at eight percent relative humidities (less than 0.001 to 100% RH) for each temperature were computed. A Bacillus cereus strain with high heat resistance was cultured and the resistance determined in phosphate buffer (D sub 121.1 = 2.16 min and z = 8.7 C). The profile of the dry heat resistance of B. megaterium is summarized and the most resistant condition to the three spores (Bacillus subtilis var. niger, ATCC 29669, and Bacillus stearothermophilus, strain 1518) is compared

Campbell, J. E.

Reyes, A. L.

English

NASA Technical Reports Server

ECOLOGY AND THERMAL INACTIVATION OF MICROBES 
'N AND ON INTERPLANETARY SPACE VEHICLE 
COHPONENTS 
Fifty- f our th Quarterly Report of ??ogrcse 
Order No. W-13411 
July 1, 1978 - September 30, 1978 
(NASA-CR-158627) ECOLCGY A N D  THERMAL N79-25705 
I N R C ' T I V A T I O N  OF HTCBOBES IN A N D  ON 
T N T E R P L A N P T A P Y  SPACE V E H I C L E  C O ~ P O N E A T S  \C 
i ? u a r t ? r l y  Progress Report, 1 Jul. 1978 - 30 U n c l a s  
S e r .  1978 (Food a n d  Drug Administration) G3/51 26866 
Conducted by 
Division of Microbiology - Cincinnati Food Research Laboratory 
Bureau of Foods 
Food and Drug Administration 
for the 
National Aeronautics and Space Administration 
Washington, D. C. 
U. S. Department of Health, Education, and Welfare 
Food and Drug Administration 
1090 Tusculum Avenue 
Cincinnati, Ohio 45226 
DECEMBER 19 78 
https://ntrs.nasa.gov/search.jsp?R=19790017534 2020-03-21T21:50:27+00:00Z
Repor t Prepa r2d by : 
ECOLOGY AND T H E W  INACTIVATION OF MICROBES 
IN AND ON IN'ERPLANETARY SPACE VEHICLE 
COMPONENTS 
Fifty-fourth Quarterly Report of  Progress 
Order No. W-13411 
July 1,  1978 - September 30, 1978 
Contributors: 
A. L .  Reyes 
A. J .  Wehby 
R .  G.  Crawford 
J .  T.  Peeler 
J .  E. Campbell 
--- - -A_ 
A .  L. Reyes 
Report Submit tec! and Forwarded by: 
! / -  
m e ,  ~ h . 6 :  
principal Investigator 
ECOLOGY ANTI THERMAL INACTIVATION OF MICROBES 
TN AND ON INTERPLANETARY SPACE VEHICLE 
COMPONENTS 
INTRODUCTION 
The experiments conducted t o  determine the hea t  resistance of Bac i l l u s  
megaterium ATCC 6458 a t  90 and 100°C were completed. Est imates  from r e p l i c a t e  
experiments a t  e i g h t  percent  r e l a t i v e  humidi t ies  (< 0.001 t o  100% RH) f o r  each 
cemperature were computed. A Bac i l lus  cereus s t r a i n  with high hea t  r e s i s t s n c e  
was cu l tured  and the r e s i s t a n c e  determined i n  phosphate bu f f e r  (D 121.1 - 2.16 
min and z = 8 .7 ' 0 .  
This r epo r t  summarizes t he  p r o f i l e  of the  dry  hea t  r e s i s t a n c e  of B. m e g a -  
terium and a l s o  compares t he  most r e s i s t a n t  condi t ion  t o  t he  t h r ee  spores  
(Bac i l lus  s u b t i l i s  var .  n iger ,  ATCC 29669, nnd Bac i l l u s  s tearothermophilus ,  
s t r a i n  1518) previously reported (50th and 52nd Quar te r ly  Reports).  
The experimental condi t ions  f o r  B. megaterfum a r e  t he  same a s  reported i n  
the 53rd Quarter ly  Report. 
I. EXPERIMENTAL 
A. Test  organism 
B. megaterium ATCC 6458 was obtained from the  American Type Culture  Col- 
- 
l e c t i o n  (ATCC), Rockville,  Md. 
B. Spore p r e ~ a r a t i o n  
An inoculum of a c t i v e l y  growing B. megaterium c e l l s  was prepared from a 
harvested spore crop. Spore crops were then produced according t o  Angelo t t i  
e t  a l .  (1 ) .  Spore suspensions were washed f i v e  t imes, pooled, and the  s tock  
suspension was s to red  i n  double d i s t i l l e d  water a t  5OC u n t i l  use. 
C. Determination of dry hea t  r e s i s t a n c e  
The closed can system (19th Quarter ly  Report) was used f o r  the  dry hea t  
i n a c t i v a t i o n  s tud i e s .  Duplicate cups were inoculated wi th  a micropipet te  i n  
6 
0.01 m l  amounts t o  give about 1 x 10 spores  per  cup. The inocula ted  cups,  
cans,  l i d s ,  and conten ts  were d r i e d  i n  a vacuum oven f o r  PO0 mia a t  45 to 50% 
(1.5-inch Hg pressure  ab solute)  . 
An increased  drying e f f i c i e n c y  was achieved by purging the  oven wi th  dry 
n i t rogen  every 10 min f o r  t he  f i r s t  90 min. This was followed by f i v e  coneecu- 
t i v e  purges of n i t rogen ,  with a  vacuum cyc l e  between each purge. Af t e r  c5e 
drying process,  the cans,  l i d s ,  and conten ts  were removed from the  oven and 
cooled t o  about  30°C i n  a n  e q u i l i b r a t i o n  hood overnight .  An amount of  water 
o r  dess ican t  (P205) was placed i n  the can t o  achieve the  des i red  X RH a t  ee- 
l ec t ed  temperatures. Tie cans were sea led  and removed from the  hood, and the  
seams of each can soldered.  Cans t h a t  were prepared t o  y i e l d  a  s e l e c t e d  X RH 
a t  a p a r t i c u l a r  temperature were completely immersed i n  a  s i l i c o n e  o i l  ba th  
opera t ing  a t  the des i red  test temperature (+O.l°C). Cans were withdrawn a t  
- 
des i red  t i n e  i n t e r v a l s  and plunged immediately i n t o  a  r e f r i g e r a t e d  water ba th  
( 5 O C )  f o r  15 min. The cans were dr ied  wi th  s teri le towels and opened with an 
e l e c t r i c  can opener. Sample cups were placed i n  tubes of peptone water  and 
soni f fed .  P l a t e  counts were nude f o r  each sample us ing  f o r t i f i e d  t r y p t i c a s e  
soy agar (0.1% so lub le  s t a r c h  and 0.2% yeas t  e x t r a c t )  a s  the recovery medium. 
P l a t e s  were incubated f o r  2 days i n  a  3S°C incubator  p r i o r  t o  counting. 
D. Determination of wet hea t  r e s i s t a n c e  
The spore  suspension was d i l u t e d  i n  0.067 M phosphate b u f f e r  (pH 7.0) t o  
6 give approximately 1 x 10 spores  per  m l .  Two m i l l i l i t e r s  of t h i s  inoculum was 
d ~ s p e n s e d  i n t o  each of 13  x 100-nun b o r o s i l i c a t e  g l a s s  tubes and flamed sea led .  
The tubes were placed i n  metal racks and submerged completely i n  a  constant- 
temperature o i l  bath.  A l l  thermal i n a c t i v a t i o n  determinat ions were made i n  
dup l i ca t e  tubes. Af te r  each hea t ing  i n t e r v a l ,  t h e  tubes were immediately 
immersed i n  a S°C-refrigerated water ba th  f o r  10 adn. Samplee were aeeayed by 
the  pour p l a t e  method us ing  f o r t i f i e d  t r y p d c a s e  soy agar.  P l a t e  counte  were 
obtained a f t e r  2 days' incubat ion a t  3S°C. 
E. S t a t i s t i c a l  c a l cu l a t i one  
D values  were est imated f o r  the wet hea t  runs by computing t h e  l i n e a r  
regress ion  c c  l o g  surv ivors  versus  time a t  each temperature. The D va lue  is 
10 
the  abso lu te  value of t he  inverse  slope. A value  t h a t  es t imatee  t h e  t ime t o  
reduce t he  i n i t i a l  population 99.99% (F value)  was ca l cu l a t ed  f o r  each experiment i n  t h e  
closed can system. Since the  i n i t i a l  concent ra t ions  were no t  cons tan t ,  t he  end- 
point  was computed using the  f r a c t i o n  su rv iv ing  versus  t i m e .  The F va lue  was 
determined by l i n e a r  i n t e r p o l a t i o n  about 0.0001 and cor rec ted  f o r  come-up time. 
11. RESULTS AND DlSCUSSION 
The hea t  r e s i s t a n c e  of B. megacerium ATCC 6458 was observed a t  e i g h t  per- 
cen t  r e l a t i v e  humidi t ies  ( ~ 0 . 0 0 1 ,  0.06, 1.2, 6.7, 29.1, 49.5, 69.3, and 100% RH) 
f o r  90°C and (<0.001, 0.04, 1.6, 7.1, 31.7, 51.5, 71.3, and 100% RH) fo r  100°C. 
Estimates of the  t i m e  (h) t o  reduce t he  i n i t i a l  populat ion 99.99% (F value) a r e  
shown I n  Table 1. The F values  determined f o r  110°C were reported i n  t he  53rd 
Quarter ly  Report. Percent c o e f f i c i e n t s  of v a r i a t i o n  computed from r e p l i c a t e  
observat ions ranged from 0.8 t o  19.2 (90 and 100°C d a t a ) .  The maximum resis- 
t a r c e  occurred a t  30% RH. 
A p lo t  of the  mean F values  versus %RH is shown i n  Fig. 1 f o r  a l l  t h r e e  
temperatures.  Each mean was connected by a l 'ne .  The ind i J idua1  es t imates  
were f i t t e d  t o  a l i n e a r  regress ion  of l og  F versus  temperatl.lre (OC) , and 10 
the abso lu te  value of the inverse  s l o p e  was ca lcu la ted .  This  is the  z value  
given i n  Table 2. The z values  ranged from 15 t o  30.5. 
Plots  of the average F values (11)  and the  est imated regreosion l i n e e  are 
shown i n  Fig. 2 f o r  four t y p i c a l  XRH. The r e s i s t ance  ofB.  megaterium ATCC 6458 
spores  is about the same f o r  < O . U O l X  RH (P205) and 100% W. However, a t  10O0C the  
30% RH condit ion produced r e s i s t ances  more than 20 times those observed a t  <0,001 
and 100% RH. 
The p a t t e r n  of  r e s i s t ance  is s i m i l a r  f o r  a l l  the  spores  examined t o  d a t e  
(11. s u b t i l i s  var .  a e z ,  ATCC 29669, - B. stearothermophilus,  and - 8. mgater ium 
ATCC 6458). The a rea  of h ighes t  r e s i s t ance  has been observed extending from 
1% RH t o  about 50% RH. The peak r e s i s t ance  f o r  the f i r s t  two spec ies  waa 
about 10% RH, with 30% RH being the most r e s i s t a n t  condi t ion  f o r  t h e  l a t t e r  two. 
A p lo t  of the most r e s i s t a n t  condi t ions f o r  spores  of the four  spec ies  
s tud ied  t o  da t e  is shown i n  Fig. 3. The dot ted  l i n e s  represent  an  extrapola-  
t i o n  outs ide  of the range of experimental condi t ions.  When compared a t  100°C, 
the F values range from 25 h f o r  g. megaterium ATCC 6458 (30% RH), the l e a s t  
r e s i s t a n t  of the four  spec ies ,  t o  720 h f o r  ATCC 29669 (7% RH), the  most r e s i s -  
t an t  spec ies .  At t h i s  temperature i n  the  closed can system, the  g r e a t e s t  
r e s i s t ance  of ATCC 29669 was almost 30-fold higher  than t h a t  of - B. megaterium. 
The r e l a t i v e  order  of hea t  r e s i s t ance  was d i f f e r e n t  when spores  of these  
spec ies  were s tudied  i n  wet hea t  condi t ions i n  phosphate buffer .  1. s tearo-  
thermophilus was then the most r e s i s t a n t  s t r a i n .  
- 
111. FUTURE STLQIES 
The est imates  of r e s i s t ance  f o r  1. cereus i n  phosphate bu f fe r  w i l l  be 
reported along with r e s u l t s  froru 120°C f o r  e i g n t  percent  r e l ae l  ve humidi t ies .  
REFERENCE 
1.  Angelotti ,  R . ,  J .  H .  Maryaneki, T. P. Butler, J .  T. Peeler,  and J.  E. 
Campbell. 1968. Influence of  spore moisture content on the dry-hut  
resistance o f  Baci l lus  e u b t i l i e  r a t .  niger.  Appl. Microbiol. 16:735-745. 
Table 1. Thermal resistance of B. megaterim ATCC 6458 
at eight percent relative humiditer ( X  RH) 
Temp. Time to reduce Mean X coefficient 
('C) X RH N 99.99% (h) (h) of variation 0 
Table 2. Sununary of z values over three temperatures 
P 
(90, 100, llO°C) for eight percent relative humidities ( X  U) 
B. amgaterium ATCC 6458 
- 
Correlation 
coefficient 
( r) 
< 0.001 
0.04 
1.4 
6.9 
29.9 
50.9 
69.8 
100.0 
Average 
% CV 
Fig. 1, Relationship of % RH to the time (h) to reduce an initial 
population of B .  megaterium ATCC 6458 99.99% at three 
temperaturea (90, 100, and llO°C). 

Fig. 2. Relation of the  time (h) to reduce an in i t i a l  popclation 
of B. megaterium ATCC 6458 99.99% versus teqerature ('C) . 
- 
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Fig.  3.  Relation of the maximum t i m e  (h) to reduce an i n i t i a l  
population of four spores 99.99% versus temperature ("C) . 
TEMPERATURE ( O C )  


Ecology and thermal inactivation of microbes in and on interplanetary space vehicle components

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